Binding Mode and Structure–Activity Relationships of ITE as an Aryl Hydrocarbon Receptor (AhR) Agonist

Article abstract of DOI:10.1002/cmdc.201700669

Discovered as a modulator of the toxic response to environmental pollutants, aryl hydrocarbon receptor (AhR) has recently gained attention for its involvement in various physiological and pathological pathways. AhR is a ligand-dependent transcription factor activated by a large array of chemical compounds, which include metabolites of l-tryptophan (l-Trp) catabolism as endogenous ligands of the receptor. Among these, 2-(1′H-indole-3′-carbonyl)thiazole-4-carboxylic acid methyl ester (ITE) has attracted interest in the scientific community, being endowed with nontoxic, immunomodulatory, and anticancer AhR-mediated functions. So far, no information about the binding mode and interactions of ITE with AhR is available. In this study, we used docking and molecular dynamics to propose a putative binding mode of ITE into the ligand binding pocket of AhR. Mutagenesis studies were then instrumental in validating the proposed binding mode, identifying His 285 and Tyr 316 as important key residues for ligand-dependent receptor activation. Finally, a set of ITE analogues was synthesized and tested to further probe molecular interactions of ITE to AhR and characterize the relevance of specific functional groups in the chemical structure for receptor activity.

Full text of DOI:10.1002/cmdc.201700669

Accepted Article
Title: Binding Mode and Structure-Activity Relationships of ITE as Aryl
Hydrocarbon Receptor (AhR) Agonist.
Authors: Daniela Dolciami, Marco Gargaro, Bruno Cerra, Giulia Scalisi,
Luana Bagnoli, Giuseppe Servillo, Maria Agnese Della Fazia,
Paolo Puccetti, Francisco J Quintana, Francesca Fallarino,
and Antonio Macchiarulo
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ChemMedChem
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Binding Mode and Structure-Activity Relationships of ITE as Aryl
Hydrocarbon Receptor (AhR) Agonist.
Dr. Daniela Dolciami,[a, e] Dr. Marco Gargaro,[b, e] Dr. Bruno Cerra, Dr. Giulia Scalisi, Dr. Luana
[a]
[b]
[
a]
[b]
[b]
[b]
Bagnoli, Dr. Giuseppe Servillo, Dr. Maria Agnese Della Fazia, Prof. Paolo Puccetti, Prof.
Francisco J. Quintana,[c, d] Prof. Francesca Fallarino, Prof. Antonio Macchiarulo.*
[b]
[a]
[
a]
Dr. Daniela Dolciami, Dr. Bruno Cerra, Dr. Luana Bagnoli, Prof. Antonio Macchiarulo.
Department of Pharmaceutical Sciences,
University of Perugia,
via del Liceo, 1 - 06123, Perugia (Italy).
E-mail: antonio.macchiarulo@unipg.it
[
[
b]
c]
Dr. Marco Gargaro, Dr. Giulia Scalisi, Dr. Giuseppe Servillo, Dr. Maria Agnese Della Fazia, Prof. Paolo Puccetti, Prof. Francesca Fallarino,
Department of Experimental Medicine,
University of Perugia,
via Gambuli, 1 – 06132, Perugia (Italy).
Prof. Francisco J. Quintana,
Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital,
Harvard Medical School,
Boston (USA).
[
[
d]
e]
Prof. Francisco J. Quintana,
The Broad Institute of Harvard and MIT,
Cambridge (USA).
These authors contributed equally to the work.
Supporting information for this article is given via a link at the end of the document.
Abstract: Discovered as modulator of the toxic response to
environmental pollutants, aryl hydrocarbon receptor (AhR) has
recently gained attention for its involvement in various physiological
and pathological pathways. AhR is a ligand-dependent transcription
factor activated by a large array of chemical compounds, which
include metabolites of L-Tryptophan (L-Trp) catabolism as
endogenous ligands of the receptor. Among these, 2-(1’H-indole-3’-
carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is attracting
interest in the scientific community, being endowed with non-toxic,
immunomodulatory and anticancer AhR-mediated functions. So far,
no information about the binding mode and interactions of ITE to AhR
is available. In this study, we used docking and molecular dynamics
to propose a putative binding mode of ITE into the ligand binding
pocket of AhR. Mutagenesis studies were then instrumental to
validate the proposed binding mode, identifying His285 and Tyr316
as important key residues for ligand-dependent receptor activation.
Finally, a set of ITE analogues was synthesized and tested to further
probe molecular interactions of ITE to AhR, and characterize the
relevance of specific functional groups in the chemical structure for
receptor activity.
aromatic hydrocarbons (PAHs), act as AhR agonists. Recently,
some natural products have also been characterized as AhR
ligands, including the bacterial-derived metabolite 1,4-dihydroxy-
3
2-naphthoic acid (1,4-DHNA, 2), and products of L-Tryptophan
(L-Trp) catabolism comprising L-Kynurenine (L-Kyn, 3), ICZ
(indolo[3,2-b]carbazole, 4), FICZ (6-formylindolo[3,2-b]carbazole,
5), malassezin (6), and ITE (2-(1’H-indole-3’-carbonyl)-thiazole-4-
carboxylic acid methyl ester, 7) (Figure 1).4-7 In particular, ITE (7)
was isolated from porcine lung in 2002 and it has been reported
as a high affinity AhR ligand (Ki = 3nM) being able to compete
3
with H-TCDD for binding to human, murine, zebrafish and killifish
isoforms of the receptor. The agonistic activity of 7 is blocked by
the AhR antagonist 3’-methoxy-4’-nitroflavone (8; Figure 1).6
1
5
2
3
7
4
Introduction
6
8
Aryl hydrocarbon Receptor (AhR) is
a
ligand-activated
Figure 1. AhR modulators. TCDD, 1; 1,4-DHNA, 2; L-Kynurenine, 3; ICZ, 4,
FICZ, 5; malassezin, 6; ITE, 7; 3’methoxy-4’-nitroflavone, 8.
transcription factor known for its involvement in human responses
to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD, 1; Figure 1). In addition to this extensively
characterized role in detoxification of xenobiotic, during the last
few years the role of AhR in other physiologically and
pathologically relevant pathways have been reported, including
regulation of stem cells, embryogenesis, immune functions,
inflammation and carcinogenesis.1,2 Many exogenous compounds
possessing different physicochemical properties, for example
halogenated aromatic hydrocarbons (HAHs) and polycyclic
Pharmacological
studies
have
pinpointed
both
immunosuppressive and anticancer functions of ITE (4) that are
mediated by the activation of AhR. Specifically, ITE (7) is able to
induce
functional
FoxP3+
Treg
cells,
leading
to
immunosuppressive effects in
a
model of experimental
autoimmune encephalomyelitis (EAE).8 Immunomodulatory
effects of ITE (7) have also been observed in a model of
1
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ChemMedChem
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experimental autoimmune uveitis (EAU) in response to Results and Discussion
9
uveitogenic antigen. Moreover, ITE (5)-induced AhR activation
has been reported to inhibit the expression of co-stimulatory
molecules and secretion of cytokines by dendritic cells (DCs) from
mice, and also from Behcet’s disease (DRE) patients.10 Likewise,
AhR activation has been shown to inhibit Th17 inflammatory
response, regulating both DCs and CD4(+) T cells derived from
patients with allergic rhinitis.11 Besides affecting differentiation
and functions of Th17/Treg cells, additional AhR mediated
immunological effects of ITE (7) have emerged, specifically the
ability of the compound to suppress B cells differentiation into Ig-
secreting plasma cells.12 AhR mediated anticancer activities of
ITE (7) have also been reported including the suppression of
proliferation and migration of ovarian cancer cells,13 and the
reduction of the tumorigenic potential of stem-like cancer cells in
orthotopic xenograft tumor models.14
Docking Study and Molecular Dynamics. Docking studies and MD
simulations were instrumental to characterize the interaction
pattern of ITE (7) to PAS-B domain of AhR. Using our previously
generated homology models of murine PAS-B AhR (a-d),20 ITE
(7) was docked into each of the four models. Briefly, the four
models of PAS-B AhR feature different properties of the ligand
binding cleft. The ligand binding cavities of model a and c have a
small size with hydrophobic properties; the binding clefts of model
b and d are large and display hydrophilic properties. Hence, each
of the four models of PAS-B AhR represents a specific
conformation of the ligand binding cavity that may accommodate
ligands with different physicochemical properties. In order to
improve the reliability of the energy scoring function by better
considering dispersion forces of hydrophobic and/or π-π stacking
interactions, docking studies were conducted using a method
incorporating quantum mechanical/molecular mechanical
(QM/MM) calculations.21
As a result, ITE (7) shows the best energy score (XP-Gscore =
-8.421kcal/mol; Table S1 of supplementary material) when
docked into model d which is endowed with the most pronounced
hydrophilic features. This docked pose shows a hydrogen bond
mediated engagement of Tyr316 by the carbonyl group of ITE (7),
and the presence of aromatic contacts between the thiazole
moiety and the side chain of His285 as well as hydrophobic and/or
aromatic contacts between the indole ring and side chains of
Phe318 and Tyr304 (Figure 2). It should be mentioned that
alternative binding poses may be envisaged in the remaining
docking solutions of models a-d, with RMSD > 2.0 Å from the top
scored binding pose of ITE (Table S1, Figures S1-S7).
8
Noteworthy, the above immune regulatory and anticancer
functions of ITE (7) combine with lack of significant toxicity, a
finding at odds with the toxicity of other potent AhR agonists such
as TCDD (1). Notably, ITE-activated AhR forms a transcriptionally
competent complex which binds to Dioxin Responsive Elements
(
DRE) similarly to TCCD-activated AhR, however ITE does not
1
5,16
provoke toxic effects in rat and mouse fetus.
As an attempt to provide an explanation to the divergent toxicity
of ITE (7) and TCDD (1), a recent study surprisingly pinpointed
that these ligands regulate similar immediate changes in gene
expression in mouse lung fibroblasts.17 Accordingly, ITE (7) and
TCDD (1) bind to PAS-B (PER-ARNT-SIM-B) domain of AhR,
triggering analogous conformational changes of the receptor that
lead to the same initial transcriptional response. Hence, authors
suggested that the lack of toxicity of ITE (7) may be ascribed to
its ability to form a shorter life-time complex with the receptor than
that induced by TCDD (1), resulting in different kinetics of gene
expression.17 Although no crystallographic information exists on
the structural complex between ITE (7) and AhR that may be
instrumental to infer about different binding modes of ITE (7) and
TCDD (1), previous docking studies into homology models
suggested that both ligands may adopt a similar binding mode to
PER-ARNT-SIM-B domain (PAS-B) of the receptor.18,19
PHE318
TYR304
TYR316
More recently, we have shown that TCDD (1) and two other
different L-Trp metabolites, namely L-Kyn (3) and FICZ (5), bind
to PAS-B of AhR exploiting different key interactions with distinct
set of fingerprint residues.20 The outcome is the stabilization of
different conformations of the receptor associated to the
transcription of specific target genes.
HIS285
With the aim of shedding further light on the binding mode of ITE
(
7) to AhR and assessing potential difference from TCDD (1), in
Figure 2. ITE best docking pose in model d. The most important residues for
the interaction are represented in sticks. Green lines indicate π-π stacking
interactions, whereas dashed pink line stands for H-bond.
this work we investigate the interaction of ITE (7) to PAS-B
domain by integrating an approach composed of computational
studies, mutagenesis experiments and synthesis of ITE
analogues. Specifically, in the first part of the study we have
performed docking and molecular dynamic (MD) simulations to
generate a prospective working hypothesis on the binding mode
of ITE (7) to PAS-B domain of AhR. Results instrumental for
designing mutagenesis experiments on proposed fingerprint
residues for ITE were validated by AhR-specific gene reporter
transcription assays. Finally, a set of ITE analogues were
designed, synthetized and tested to further probe molecular
interactions of ITE to AhR and characterize the relevance of
specific functional groups in the chemical structure for receptor
activity.
Although scoring functions have been generally considered
problematic at distinguishing the crystallographic conformation
from the set of docked poses,22 improvements obtained by
incorporating QM/MM calculations prompted us to select the
aforementioned top ranked pose of ITE (7) as working hypothesis
of binding mode for MD simulations, according to a criteria of best
energy score.2
3-25
This docked pose was thus submitted to six
independent runs of 50 ns MD simulations in order to assess the
stability of the interactions and identify fingerprint residues. The
inspection of the root mean square deviation (RMSD) of protein
2
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ChemMedChem
10.1002/cmdc.201700669
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backbone atoms reveals a stabilization of the complex after the
first 10 ns in all MD simulations (Figure 3).
The inspection of root mean square fluctuations of residues
(RMSF; Table S2, supplementary material) suggests that
conformations of most residues within 3Å of the ligand docked
pose are generally stable, with only Leu309, Gly315 and Tyr316
showing RMSF > 1.5 Å in at least one MD simulation. In particular,
these conformational drifts may suggest the engagement of ITE
(
7) with additional hydrophobic and aromatic interactions by the
side chains of Leu309 and Tyr316, and/or with the optimization of
hydrogen bond interactions involving the backbone atoms of such
residues.
Next, we defined putative fingerprint residues of ITE (7) as those
yielding average values of ligand interaction occupancy higher
than 0.3 in all MD trajectories. Accordingly, the following potential
fingerprint residues were identified in MD simulations: His285,
Tyr316 and Phe318 (Table S3, supplementary material).
Specifically, Phe318 makes a hydrogen bond between its
backbone amide group and ITE carbonyl group (Figures 6A-D),
that may also be stabilized by mean of a water bridge interaction
(as observed in the first MD simulation, Figure 6A).
Notwithstanding, contribution of such interactions to the agonist
activity of ITE (7) may be negligible, as suggested by indirect
evidences from structure-activity relationship studies of another
Figure 3. RMSD (Root Mean Square Deviation) values (Å) of protein backbone
atoms along molecular dynamic trajectories.
3
2.5
1st MD
2nd MD
3rd MD
4th MD
5th MD
6th MD
2
1
.5
26
1
L-Trp metabolite, namely malassezin (6).
0.5
A
B
0
10
20
30
40
50
Time (ns)
Figure 4. RMSD values (Å) of ITE atoms along molecular dynamic trajectories.
In the first simulation, the RMSD value undergoes to an increase
around the 35th ns and then remains stable until the end of the
simulation. In the fourth simulation, which is the one reaching the
highest value of RMSD, a jump around 10th ns is detectable.
Moreover, in the sixth simulation, the complex undergoes to a
RMSD jump between 15th and 25th ns and then returns to lower
drifts. To assess whether these movements affect the docked
pose, the RMSD of ITE (7) and root mean square fluctuations
C
D
(
RMSF) of residues lying in a shell of 3Å around the ligand docked
pose were calculated along the trajectories. Examining the RMSD
plot (Figure 4), significant jumps of ligand are detectable in the
fifth and sixth simulations. Specifically, the jump around the 20th
Figure 6. Crucial interactions of Phe318 with ITE (7) during MD simulations.
Phe318 is involved in Hydrogen bond with carbonyl group of ITE, in one case
ns of the fifth MD simulation is linked to rotational and translational
movements of the entire ligand within the binding pocket, as
exemplified in Figure 5A.
st
nd
mediated by a water bridge. (A) 1 MD simulation. (B) 2 MD simulation. (C)
3
rd
th
MD simulation. (D) 6 MD simulation.
Conversely, the large drifts during the first five nanoseconds of
the sixth MD are ascribed to torsional movements of the indole
moiety (Figure 5B).
Tyr316 makes a hydrogen bond between its backbone amide
group and ITE carbonyl oxygen (as observed in the first MD
simulation, Figure 7A) or with the nitrogen atom of the thiazole
ring of ITE (7) (as observed in the second and fifth MD simulations,
Figure 7B, C), being this latter also stabilized by means of water
bridges (as observed in the sixth MD simulation, Figure 7D).
His285 is involved in a π-π stacking aromatic interaction with the
thiazole ring (as observed in the second and fifth MD simulation,
Figure 8A, B) or alternatively with the indole ring of ITE (as
observed in the sixth MD simulation, Figure 8C). The same
residue makes a hydrogen bond interaction through a water
bridge with the nitrogen atom of the thiazole ring of ITE (as
observed in the sixth MD simulation, Figure 8D). Of note, no
A
B
Figure 5. (A) Rotational and translational movements of ITE (7) during the 5th
MD simulation. (B) Translational movement of ITE (7) during the 6th MD
simulation.
hydrogen bond interaction was observed between ITE (7) and
Ser359, as previously reported from other docking studies.18,19
3
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A
B
When assayed in a luciferase reporter assay using mouse DCs
H285A
Y316A
reconstituted with mutated AhR
or AhR
, ITE (7) exhibits
a complete loss of biologic activity, in terms of potency and likely
affinity, compared to AhRWT transfected counterpart (Figure 9B).
In contrast, only
a
minor and negligible decrease of
transactivation activity is observed when ITE (7) is tested in MEF
reconstituted with AhRQ377A, suggesting a slightly lower affinity of
the ligand at this mutant receptor. Hence, mutagenesis
experiments support the role of His285 and Tyr316 as fingerprint
residues of ITE (7), highlighting poor relevance of Gln377 for
ligand activity.
C
D
A
Figure 7. Crucial interactions of Tyr316 with ITE (7) during MD simulations.
Tyr316 is involved in Hydrogen bond with Nitrogen atom of thiazole ring, in one
st
case mediated by a water bridge. In the 1 MD, Tyr316 binds carbonyl group of
st
nd
th
ester moiety. (A) 1 MD simulation. (B) 2 MD simulation. (C) 5 MD simulation.
(
th
D) 6 MD simulation.
B
A
B
C
D
Figure 9. (A) AhR expression in MEF transfected with WT or H285A, Y316A,
G377A AhR mutants. AhR-deficient mouse embrional fibrobasts (MEF) were
transfected with WT or different AhR mutants: AhR(H285A), AhR(Q377A),
AhR(Y316A) or AhR(Y316A/Q377A). After 24 h, cells were lysed, and analysed
for AhR expression by immunoblotting, using a specific AhR antibody. β-tubulin
was used as a loading control. Data are from one experiment of three. (B)
Transactivation activity of WT, H285A, Y316A or G377A AhR by ITE (7). AhR-
deficient mouse embrional fibrobasts (MEF) were transfected with WT or
different AhR mutants:
AhR(H285A), AhR(Q377A), AhR(Y316A) or
AhR(Y316A/Q377A). After 6 h, cells were lysed, and the amount of AhR–
ARNT–DRE complex was achieved by measuring luciferase activity. Data mean
± s.d. of three independent experiments. ***p ≤ 0.001 as determined by a
Student’s t test.
Figure 8. Crucial interactions of His285 with ITE (7) during MD simulations.
His285 is mostly involved in π-π stacking interaction with thiazole or indole rings.
th
In the 6 MD, His285 interacts by means of a water bridge with Nitrogen atom
nd
th
th
of thiazole ring. (A) 2 MD simulation. (B) 5 MD simulation. (C) 6 MD
th
simulation. (D) 6 MD simulation.
Design, Synthesis and Structure-Activity Relationships of ITE
Analogues. Docking study and MD simulations have evidenced
hydrophobic contacts (Phe289, Leu309, Phe318), π-π stacking
aromatic interactions (His285, Phe289, Tyr316, Phe318), and
hydrogen bonds (His285, Thr283, Leu309, Tyr316, Phe318) as
involved in the stabilization of ITE (7) binding to PAS-B AhR.
Among the proposed fingerprint residues, mutagenesis
experiments have proved the importance of His285 and Tyr316
for the transcriptional activity of ITE (7), and likely for the affinity
of ligand to the receptor. These residues, however, are involved
in different kinds of interaction with specific pharmacophoric
groups of ITE (7), depending on the trajectory that is analyzed out
of the six MD simulations. Specifically, His285 is involved in a π-
π stacking aromatic interaction with the indole ring or the thiazole
moiety of ITE (7). A water mediated hydrogen bond is also
observed in a trajectory between His285 and the nitrogen atom of
the thiazole ring. Likewise, hydrogen bonds are observed
between the backbone atoms of Tyr316 and the ester group or
thiazole ring of the ligand.
Mutagenesis experiments. On the basis of the results of docking
study and MD simulations of ITE (7), two mutant AhR receptors
were engineered carrying His285Ala mutation (H285A) and
Tyr316Ala mutation (Y316A). It is worth noting that residue
Gln377 was formerly identified as important in mediating a key
hydrogen bond interaction with L-Kyn (3), but not involved in
interactions with TCDD (1) and FICZ (5).20 Residue Gln377 does
not fall within 3Å of ITE docked pose, and shows only a weak
hydrogen bond interaction with the ligand in one simulation (fourth
MD simulation, average occupancy value of 0.19 ± 0.20).
Notwithstanding, a third mutant receptor was designed bearing
Gln377Ala mutation (Q377A) to probe by mutagenesis the role of
such polar residue in mediating ITE (7) activity. To explore the
impact of specific amino acid residues in ITE-induced AhR
activation we established mouse embryonic fibroblast from AhR
-/-
-/-
deficient mice (MEF AhR ). Thus, MEF AhR were reconstituted
WT
H285A
, or AhRY316A, or AhRQ377A. Transfection
with AhR or AhR
efficiency was similar for the four constructs, as revealed by
western blot analysis on cell protein lysates (Figure 9A).
4
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Overall, these observations suggest that the thiazole moiety of
ITE (7) may be an important pharmacophoric group promoting key
aromatic and/or hydrogen bond interactions within the ligand
binding pocket of PAS-B AhR. To further investigate the relevance
of the thiazole moiety and its interactions to PAS-B AhR, a set of
ITE (7) analogues were designed, synthetized and tested using
the specific AhR reporter hepatic H1L1 cells (Figure 10).
Specifically, compound 9 was conceived to bear a biososteric
replacement of the thiazole ring with an oxazole ring, keeping the
aromatic feature of this moiety while increasing its polarity (clogP
material, and tested at 1.0 M and 0.1 M concentrations in
luciferase transactivation assays. As a result, they do not show
any improvement of the transcriptional activity over ITE (7),
suggesting that each of them loses key interactions to AhR with
respect to the parent compound (Figure 11).
=
1.60, table 1). In compound 10, the aromatic feature of the
central heterocycle was removed to assess the importance of the
- stacking interaction of this moiety to the receptor with respect

to compound 9.
7
9
10 11 12 13 14 15 16
9
10
14
11
15
12
16
Figure 11. Transactivation activity of AhR by ITE (7) and different ITE
derivatives (9-16). Hepatic H1L1 cells were transfected with WT or different AhR
mutants: AhR(H285A), AhR(Q377A), AhR(Y316A) or AhR(Y316A/Q377A).
After 6 h, cells were lysed, and AhR activation was evaluated by measuring
luciferase activity. Data mean ± s.d. of three independent experiments. ***p ≤
1
3
0.001, *p ≤ 0.05, Dunnett test.
Figure 10. Structure of ITE analogues (9-16), which have been tested in biologic
assays.
In particular, compound 9 is the most active analogue within the
series, albeit it shows a drop of transcriptional activity with respect
to ITE (7). This observation firstly suggests that the increase of
ligand hydrophilic property is detrimental for the transcriptional
activity. In agreement with mutagenesis data, the lack of activity
of compound 10 pinpoints the importance of π-π stacking
interactions with His285 and likely Tyr316, whereas a less
important role is ascribed to hydrogen bond interactions of the
methyl ester moiety. This observation, however, combines with
structure activity relationships of compounds 11-16 pinpointing
how π-π stacking interactions are a necessary but not sufficient
condition to bestow potency on ligands. Specifically, the low
activity observed for compounds 11-16 prompts that removal of
the methyl ester group is detrimental for ligand potency.
Noteworthy, this observation supports the presence of hydrogen
bond interactions between the methyl ester moiety of ITE (7) and
the backbone atoms of Phe318 and Tyr316. Although these
interactions were observed in MD simulations, it is not possible to
probe them by mutagenesis experiments given the lack of
involvement of side chain atoms. The better activity of compounds
Finally, compounds 11-16 were designed to further probe -
stacking interactions with PAS-B AhR, using five- or six-member
aromatic groups with different HOMO and LUMO energies (Table
1).
Table 1. Molecular descriptors (cLogP, HOMO, LUMO) calculated for ITE (7)
and its analogues (9-16).
Compound
cLogP
2.15
HOMO[a]
-0.22042
LUMO[a]
-0.09203
ITE (7)
9
1.60
1.53
3.36
3.23
2.64
2.71
2.82
1.88
-0.21720
-
-0.09443
-
10
11
12
13
14
15
16
-0.21371
-0.21082
-0.21318
-0.20653
-0.20426
-0.21073
-0.07760
-0.07131
-0.06263
-0.07315
-0.07685
-0.09035
15 and 16 in the series also suggests that the presence of a
nitrogen atom as hydrogen bond acceptor group at position 2 of
the heteroaromatic ring has a role for activity, supporting the
engagement of Tyr316 in hydrogen bond interaction as resulting
from the MD study. Conversely, no apparent relationship was
found between the activities of compounds 11-16 and their
relative HOMO and LUMO energies (Table 1).
[
a] Hartree.
Taken together, MD simulations, mutagenesis studies and
structure-activity relationships of ITE analogues suggest a
pharmacophore model of ITE interaction to PAS-B AhR that is
composed of the following key structural elements (Figure 12): (i)
a first aromatic center located on the thiazole ring (Ar-1; -
stacking interaction with His285); (ii) one hydrogen bond acceptor
Docking studies of compounds 9-16 into model d of PAS-B AhR
support the design strategy, evidencing top scored solutions with
similar docked poses to ITE (7), and located in an energy window
of 2 kcal/mol from the selected binding pose of ITE into model d
(
Figure S8, supplementary material). Compounds 9-16 were then
synthesized according to schemes reported in the supplementary
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group placed at the carbonyl ester atom (HBA-1; hydrogen bond
interaction with Tyr316); (iii) one accessory hydrogen bond
acceptor group located at the nitrogen atom of the thiazole ring
for ligand binding and potency to AhR, respectively. This
information will be instrumental to enable next ITE analogue
design and/or structure-based approaches for the development of
new AhR modulators on the way to disclose novel immune-
regulatory and anticancer therapeutic agents.
(
HBA-2; hydrogen bond interaction with Tyr316, and Gln377 to a
less extend); (iv) a second aromatic center placed on the indole
moiety (Ar-2; - stacking interaction with Phe316). This
pharmacophore model was used to revisit early docking results of
ITE (Table S1), looking for additional solutions that could provide
alternative binding modes of this ligand to PAS-B AhR. Among
alternative binding poses of models a-d (RMSD > 2.0 Å; Figures
S1-S7), none was successful in matching all of the elements of
the pharmacophoric model with simultaneous interactions
involving key residues His285 and Tyr316, supporting a good
performance of the QM/MM-based docking method in this study.
Experimental Section
Synthesis. Synthetic methodologies are reported in supplementary
material.
General methods. Melting points were determined using a Buchi 535
electrothermal apparatus and are uncorrected. NMR spectra were
recorded on a Bruker AC 400 MHz spectrometer in the indicated solvent.
Chemical shifts (δ) are reported in parts per million (ppm) and are relative
6
to CD
3
OD (3.35 ppm and 49.3 ppm) and d -DMSO (2.49 ppm and 39.7
ppm). Coupling constants (J) are expressed in Hz. The following
abbreviations are used to indicate the multiplicity: s, singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet; brs, broad signal. High resolution
mass spectra (HRMS) were recorded on Agilent 6540-UHD Accurate Mass
Q-TOF LC/MS instrument. Compounds purity (>95%) was assessed by
HPLC analysis (method not validated) using a Shimadzu (Kyoto, Japan)
LC-20A Prominence equipped with a CBM-20A communication bus
module, two LC-20AD dual piston pumps, a SPD-M20A photodiode array
detector and a Rheodyne 7725i injector (Rheodyne Inc., Cotati, CA, USA)
with a 20 mL stainless steel loop. A DAD detector was utilized for analysis.
Ar-2
HBA-2
Ar-1
HBA-1
-
1
The adopted conditions for the analysis were: flow rate 1 mL min , a
prevail C18 column (250 x 4.6 mm i.d., 5 µm) for compounds 9 and 10,
and a Grace Smart RP18 (250 x 4.6 mm i.d., 5 µm) for compounds 11-16,
2 3
using a solution of H O/CH CN as the eluent. Thin layer chromatography
Figure 12. Pharmacophoric model of ITE (7) interaction to PAS-B AhR. Key
structural elements of ligand are labelled (Ar-1; HBA-1; HBA-2; Ar-2). Key
residues of receptor are highlighted with red dashed circles.
(TLC) was performed in 60 F254 (Merck) silica gel supported on aluminium
sheets. Spots were visualized by UV detector (λ: 254 nm). Reactions
products were purified by flash chromatography on silica gel (Merck 60,
230-400 mesh). ITE (7) was purchased from Tocris Biosciences. Reagents
were purchased from Sigma Aldrich and Alfa Aesar and were used without
further purification. 2-(1'H-indol-3'-yl)-2-oxoacetyl chloride (17) was
prepared according to procedures reported in literature.27
Conclusions
AhR is a ligand dependent transcriptional factor involved in the
regulation of drug metabolism, inflammation and in several
immune functions. ITE (7) is an endogenous L-Trp metabolite that
potently binds to AhR in the nanomolar range of potency. ITE (5)
mediated activation of AhR has been associated to
immunosuppressive and anticancer functions of the compound. It
is also worth noting that, in contrast to other potent AhR ligands
such as TCDD (1), ITE (7) is not endowed with significant AhR
mediated toxic effects. The immunoregulatory and anticancer
functions of ITE (7), combined with its clean toxicological profile,
make this endogenous molecule an interesting lead compound for
the development of potent and safer AhR agonists for novel
therapeutic opportunities in autoimmune disorders and cancer
disease. In this paper, we have applied an integrated
computational, genetic and synthetic approach to get insights into
the binding mode of ITE (7) to AhR PAS-B. Results highlighted
fingerprint residues of the receptor involved in binding and activity
of ITE (7), as well as identifying the thiazole ring as key
pharmacophoric element of the ligand structure. Collectively,
results of docking studies, MD simulations and mutagenesis
experiments are consistent with structure–activity relationships
obtained from a series of ITE analogues (9-16), supporting the
experimental validity of the proposed binding mode of ITE (7) to
AhR PAS-B. They pinpoint that - stacking interactions rather
than hydrophobic contacts, and specific hydrogen bonds of the
central heteroaromatic ring and methyl ester moiety are important
(
±)-Methyl
hydroxypropanoate (18).
methyl ester hydrochloride (2 mmol, 0.31 g) in CH
Et N (2 mmol, 0.27 mL) was added. After the complete dissolution, 2-(1’H-
2-[2’-(1’’H-indol-3’’-yl)-2’-oxoacetamido]-3-
28
To a stirring suspension of (D, L)-serine
Cl (45 mL) at 0 °C,
2
2
3
indol-3’-yl)-2-oxoacetyl chloride (17) (1.4 mmol, 0.3 g) was added at 0 °C
and the resulting solution turns from white to yellow. The mixture was
allowed to warm to room temperature and stirred for 12 h. Reaction
progress was monitored by TLC (eluent: CH
40 mL) was added and the mixture was extracted with CH
The combined organic extracts were dried over anhydrous Na
concentrated under reduced pressure. The crude was purified by flash
chromatography on silica gel (eluent: CH Cl /MeOH 99:1, v/v) affording
2
Cl
2
/MeOH 98:2, v/v). Water
Cl (3 x 10 mL).
SO and
(
2
2
2
4
2
2
the title compound (±)-methyl 2-[2’-(1’’H-indol-3’’-yl)-2’-oxoacetamido]-3-
hydroxypropanoate (18) (460 mg, 1.59 mmol, 80% yield) as a white solid
1
(
m.p.: 174-177 ) H NMR (CD
3
OD, 400 MHz): δ 8.84 (s, 1H), 8.33-8.35 (m,
1
(
3
1
H), 7.50-7.52 (m, 1H), 7.28-7.30 (m, 2H), 4.67 (t, J= 3.76 Hz, 1H), 4.05
dd, J = 4.0 Hz, J =11.3, 1H), 3.95 (dd, J = 3.5, J = 11.3 Hz, 1H), 3.82 (s,
H). 13C NMR (CD
OD, 100.6 MHz): δ 180.3, 170.4, 163.6, 138.4, 136.5
26.5, 123.5, 122.5, 121.6, 112.5, 111.7, 61.3, 54.6, 51.6. HPLC residence
): 6.2 min (eluent: H O/CH CN, 60:40, v/v).
1
2
1
2
3
time (t
R
2
3
(
±)-Methyl
carboxylate (10). To a suspension of 18 (0.7 mmol, 0.2 g) in freshly
distilled CH Cl (5 mL), diethylaminosulfurtrifluoride (DAST, 1.5 mmol, 0.2
2-(1’H-indole-3’-carbonyl)-4,5-dihydrooxazole-4-
29
2
2
mL) was added at -78 °C under argon atmosphere. The resulting mixture
was allowed to warm to r.t. and stirred for 6 h. Reaction progress was
2 2 2 3
monitored by TLC (eluent: CH Cl /MeOH 98:2, v/v). Anhydrous K CO (0.5
mmol, 0.14 g) was added to reaction mixture. After 30 minutes, the
6
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201700669
FULL PAPER
reaction was poured into aqueous saturated solution of NaHCO
3
(20 mL)
2H), 7.07 (s, 1H), 7.01 (s, 1H), 6.22-6.24 (m, 1H). 13C NMR (CD
OD, 100.6
3
and the biphasic mixture was extracted with CH
anhydrous Na SO and concentrated under reduced pressure. The crude
was purified by flash chromatography on silica gel (eluent: CH Cl /MeOH
9:1, v/v) affording the title compound (±)-methyl 2-(1’H-indole-3’-
2
Cl
2
(3 x 15 mL), dried over
MHz): δ 180.6, 136.8, 132.2, 132.1, 126.5, 123.8, 122.8, 121.2 (2x), 116.0,
115.5, 111.4, 109.5.
2
4
2
2
9
Furan-2-yl(1H-indol-3-yl)methanone (14).33 The title compound was
isolated (23 mg, 0.11 mmol, 11% yield) as a white solid (m.p.: 179-182 °C).
carbonyl)-4,5-dihydrooxazole-4-carboxylate (10) (130 mg, 0.48 mmol,
6
6
9% yield) as a white solid (m.p.: 214-220 °C). 1H NMR (d -DMSO, 400
CN, 60:40, v/v). 1
HPLC residence time (t
R
): 8.2 min (eluent: H
2
O/CH
3
H
MHz): δ 12.32 (brs, 1H), 8.59 (s, 1H), 8.19-8.21 (m, 1H), 7.55-7.57 (m, 1H),
6
NMR (d -DMSO, 400 MHz): δ 12.14 (brs, 1H), 8.52 (brs, 1H), 8.31 (d, J=
7
3
1
.26-7.32 (m, 2H), 5.12 (dd, J
.76 (s, 3H). 13C NMR (CD
OD, 100.6 MHz): δ 176.4, 171.4, 162.5, 139.1,
37.1, 126.1, 124.2, 123.2, 121.7, 114.4, 113.1, 70.0, 68.7, 52.9. HPLC
1 2
= 4.1 Hz, J =10.3, 1H), 4.56-4.66 (m, 2H),
7
7
1
1
.31 Hz, 1H), 8.00 (s, 1H), 7.53 (d, J= 7.82 Hz, 1H), 7.36-7.37 (m, 1H),
.22-7.26 (m, 2H), 6.74 (s, 1H). 13C NMR (CD
OD, 100 MHz): δ 177.3,
53.9, 145.7, 136.6, 134.8, 126.6, 123.2, 122.0, 121.6, 116.4, 114.3, 111.7,
11.5.
3
3
residence time (t
R
): 9.1 min (eluent: H
2
O/CH
3
CN, 60:40, v/v). FT-IR (KBr):
-
ν 3181, 3077, 2924, 1737, 1605, 1507, 1445, 1380, 1257, 1210, 1033 cm
1. HRMS (ESI) m/z [M+H]+ calcd for C14
73.0882;  = 4 ppm.
13 2 4
H N O 273.0870; found
(
1H-indol-3-yl)(pyridin-2-yl)Methanone (15).33 The title compound was
isolated (22 mg, 0.10 mmol, 10% yield) as a white solid (m.p.: 188-190 °C).
2
HPLC residence time (t
NMR (d -DMSO, 400 MHz): δ 12.11 (brs, 1H), 8.82 (s, 1H), 7.62-7.63 (m,
R
): 13.7 min (eluent: H
2
O/CH
3
CN, 70:30, v/v). 1
H
Methyl 2-(1’H-indole-3’-carbonyl)-oxazole-4-carboxylate (9).30 To a
solution of (±)-methyl 2-(1’H-indole-3’-carbonyl)-4,5-dihydrooxazole-4-
6
1
(
H), 8.36-8.38 (m, 1H), 8.03-8.05 (m, 2H), 7.61-7.64 (m, 1H), 7.50-7.54
m, 1H), 7.24-7.27 (m, 2H). 13C NMR (CD
OD, 100.6 MHz): δ 187.9, 156.7,
carboxylate (10) (0.11 mmol, 0.03 g) in CH
.14 g) was added at room temperature. The resulting suspension was
stirred for 3 h at room temperature and then concentrated. Reaction
progress was monitored by TLC (eluent: CH Cl /MeOH 98:2, v/v). The
crude was concentrated under reduced pressure and the residue was
purified by flash chromatography on silica gel (eluent: CH Cl /MeOH 99:1,
2 2 2
Cl (25 mL), MnO (1.65 mmol,
3
0
1
1
48.1, 137.9, 137.3, 136.6, 126.9, 125.7, 123.1, 123.0, 122.1, 121.7, 114.5,
11.5.
2
2
(
1H-indol-3-yl)(pyrazin-2-yl)Methanone (16). The title compound was
2
2
isolated (20 mg, 0.09 mmol, 9% yield) as a white solid (m.p.: 212 °C dec.).
HPLC residence time (t
v/v) affording the title compound methyl 2-(1’H-indole-3’-carbonyl)-
oxazole-4-carboxylate (7) (18 mg, 0.067 mmol, 60% yield) as a white solid
CN, 70:30, v/v). 1
R
): 11.0 min (eluent: H
2
O/CH
3
H
6
NMR (d -DMSO, 400 MHz): δ 12.18 (brs, 1H), 9.15 (brs, 1H), 8.67-8.84
(m.p.: 250 °C dec.). HPLC residence time (t
R
): 14.6 min (eluent:
O/CH CN, 60:40, v/v). H NMR (d -DMSO, 400 MHz): δ 12.37 (br, 1H),
m, 3H), 8.33 (brs, 1H), 7.52 (brs, 1H), 7.25 (m, 2H). 13C NMR (d -DMSO,
6
(
1
6
H
2 3
100.6 MHz): δ 185.4, 151.3, 147.2, 144.8, 143.8, 138.6, 136.7, 127.0,
9
(
.12 (s, 1H), 8.91 (s, 1H), 8.28-8.30 (m, 1H), 7.59-7.60 (m, 1H), 7.29-7.31
123.8, 122.9, 122.0, 114.2, 112.8.
m, 2H), 3.89 (s, 3H). 13C NMR (d -DMSO, 100.6 MHz): δ 182.3, 181.9,
6
1
70.9, 164.8, 164.5, 139.5, 137.1, 127.0, 124.4, 123.5, 122.1, 113.4, 112.9,
-
1
Molecular Modelling.
53.2. FT-IR (KBr): ν 3238, 1739, 1608, 1505, 1415, 1231, 1138 cm .
+
HRMS (ESI) m/z [M+H] calcd for C14
H N O
11 2 4
271.0713; found 271.0725;
Docking Study and Molecular Dynamics. The chemical structures of ITE

= 4 ppm.
(
7) and analogues (9-16) were generated using LigPrep 3.2 (Schrödinger,
General procedure for the synthesis of compounds 11-16.31 To a
mixture of indole (19) (1 mmol) in dioxane/AcOH/H O (50:20:30, v/v/v, 2
(5 mol%), bipyridine (5 mol%) and the
corresponding nitrile derivative (1.2 mmol) were sequentially added and
the resulting mixture was stirred at 100 °C for 24 h. The reaction mixture
LLC, New York, NY, 2014). For each ligand, we considered different
tautomeric and ionization states at the physiological pH of 7.0 ± 2
employing Epik 3.0 (Schrödinger, LLC, New York, NY, 2014). The
geometry of the compounds was refined using DFT/6-31G** calculations
with Jaguar 8.6 (Schrödinger, LLC, New York, NY, 2014).34 Docking
studies into the four models of PAS-B AhR were performed employing the
quantum mechanics-polarized ligand docking (QPLD) workflow and Glide
2
-
1
mL mmol ), Pd(OAc)
2
was neutralized by adding aqueous saturated solution of NaHCO
and extracted with EtOAc (3 x 15 mL). The combined organic extracts were
washed with H O (20 mL), brine (20 mL), dried over anhydrous Na SO
4
3
(20 mL)
6
.5 (Schrödinger, LLC, New York, NY, 2014).21 According to this workflow,
2
2
ligand atomic partial charges are calculated using a QM/MM approach,
with the B3LYP method and the 6-31G* basis set, and fitting the
electrostatic potential (ESP). Docking study of ITE (7) and analogues 9-16
were carried out into previously reported homology models of murine PAS-
B AhR (models a, b, c and d for compound 7; model d for compounds 9-
and concentrated under reduced pressure. The crude was purified by flash
chromatography on silica gel (eluent: n-hexane/EtOAc from 100:0 to 60:40,
v/v) affording the title compounds 11-16.
(
1H-indol-3-yl)(phenyl)Methanone (11).31 The title compound was
isolated (49 mg, 0.22 mmol, 22% yield) as brownish solid (m.p.: 168-
6),35 employing Glide extra precision mode (XP). The best scored pose
1
of ITE (7) obtained by comparing docking solutions among the four in
house homology models was selected for multiple molecular dynamic
1
72 °C). HPLC residence time (t
R
): 7.0 min (eluent: H
2
O/CH
3
CN, 1:1, v/v).
1
6
H NMR (d -DMSO, 400 MHz): δ 12.09 (brs, 1H), 8.25-8.27 (d, J=6.84 Hz,
(MD) simulations. Specifically, six MD simulations were carried out using
1
2
1
H), 7.94 (s, 1H), 7.79 (d, J=7.5 Hz, 2H), 7.52-7.62 (m, 4H), 7.22-7.28 (m,
H). 13C NMR (d -DMSO, 100.6 MHz): δ 190.4, 141.0, 137.2, 136.3, 131.5,
28.8 (2x), 126.7, 123.6, 122.4, 121.9, 115.4, 112.7.
a production time of 50 ns. Briefly, the selected PAS-B AhR ligand bound
complex from QPLD docking studies was solvated in an orthorhombic box
using TIP3P water molecules, extended 10 Å away from any protein atom.
The resulting system was neutralized by adding sodium and chlorine ions
at a concentration of 0.15 M. Periodic boundary conditions were applied to
avoid finite-size effects. Atomic partial charges of ITE were maintained as
6
(
1H-indol-3-yl)(thiophen-2-yl)Methanone (12).32 The title compound was
isolated (45 mg, 0.20 mmol, 20% yield) as a white solid (m.p.: 180-182 °C).
HPLC residence time (t
NMR (d -DMSO, 400 MHz): δ 12.14 (brs, 1H), 8.35 (s, 1H), 8.20 (s, 1H),
7
1
1
R
): 11.5 min (eluent: H
2
O/CH
3
CN, 60:40, v/v). 1
H
obtained from QPLD calculation. MD simulations were performed using
Desmond 4.0 (Schrödinger, LLC, New York, NY, 2014)28 and the OPLS-
6
.93 (s, 2H), 7.52 (d, J= 7.37 Hz, 1H), 7.22-7.26 (m, 3H). 13C NMR (CD
OD,
3
00.6 MHz): δ 182.7, 144.9, 137.0, 134.0, 131.6, 131.5, 127.5, 126.4,
23.2, 121.8, 121.4, 115.4, 111.6.
2
005 force field. The simulation protocol included starting relaxation steps
20
and a final production phase of 50 ns, as previously reported. The
occupancy of intermolecular hydrogen bonds, aromatic interactions and
hydrophobic contacts was calculated along the last 48 ns of the production
phase of each MD simulation, using a cut-off value of 30% and the
Simulation Interaction Diagram Tools implemented in Maestro 10.0
(Schrödinger Release 2014-4: Maestro, Schrödinger, LLC, New York, NY,
2014).
(
1H-indol-3-yl)(1H-pyrrol-2-yl)Methanone (13).33 The title compound
was isolated (15 mg, 0.07 mmol, 7% yield) as a reddish-brownish solid
(
H
m.p.: 225-227 °C). HPLC residence time (t
O/CH CN, 70:30, v/v). H NMR (d -DMSO, 400 MHz): δ 11.90 (brs, 1H),
2 3
R
): 12.9 min (eluent:
1
6
11.75 (brs, 1H), 8.22-8.25 (m, 2H), 7.49 (d, J= 7.70 Hz, 1H), 7.12-7.23 (m.
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201700669
FULL PAPER
Molecular Descriptors. Predicted octanol/water partition coefficient
Supporting Information: Table S1. Top ten ranked poses of ITE (7) into the
homology models a-d of PAS-B AhR; Table S2. Root mean square
fluctuations (RMSF, Å) of residues lying in a 3Å shell around ITE (7); Table
S3. Occupancy values (mean  standard deviation) of interactions
observed in the six MD trajectories between ITE (7) and binding site
residues; Figure S1. Interaction plots of top scored binding poses of ITE
(7) in models a-d; Figures S2-S7. Interaction plots of other scored binding
poses of ITE (7) in models a-d showing RMSD > 2.0Å; Figure S8. Top
scored binding poses of ITE analogues (9-16) as docked into model d, and
superimposed to the top scored binding pose ITE (7) in model d; Synthetic
methodologies; Spectroscopic and analytical characterization of
compounds 9-16, 18.
(cLogP) was calculated with QikProp (Schrödinger Release 2014-4:
Maestro, Schrödinger, LLC, New York, NY, 2014), using default options.
HOMO and LUMO energies were computed using Jaguar (Schrödinger
Release 2014-4: Maestro, Schrödinger, LLC, New York, NY, 2014) Single
Point Energy (SPE). Hartree Fock theory, accurate level of SCF were
employed to calculate Atomic Fukui Indices property.
Biology
Mouse embryonic fibroblast derivation. The mouse embryonic fibroblasts
(
MEFs) were obtained according to the guidelines of University of Perugia
Ethical Committee and the European Communities Council Directive
010/63/EU. MEFs and AhR−/− MEFs were prepared respectively from
2
C57BL/6 mice and AhR −/− C57BL/6 mice, and the preparation protocol
was adapted from J. Xu.36 The pregnant mice were sacrificed at 13,5 d.p.c.
Acknowledgements
(
day post-coitum) by cervical dislocation. 5–7 embryos can be expected
This study was also supported by Telethon Research Grant
GGP14042 to F.F.
from each pregnant female, and they should yield enough MEFs for
several experiments. The embryos were separated from placenta and
membranes and placed in 10-cm culture dishes in sterile PBS 1X. Then
liver, heart and brain were removed and discarded. The remaining part of
each embryos were washed and minced with cool razor blades and
incubated 20 minutes at 37°C with trypsin-EDTA 500 mg/L. The minced
tissues were chopped by repeated pipetting, then cell suspension were
plate in 10-cm tissue culture dishes and 10 ml of DMEM medium
containing 10% FBS (Euroclone) were added. Electroporation was used
to transfect MEFs.
Keywords: Immunotherapy • Cancer • Tryptophan • AhR • ITE
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[
[
[
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–
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
Ar-2
HBA-2
Ar-1
HBA-1
ITE is an endogenous product of L-Tryptophan catabolism. It is also a non-toxic AhR agonist endowed with immunomodulatory
properties. In this study, we used docking and molecular dynamics to investigate the binding mode of ITE into the ligand binding pocket
of AhR. Mutagenesis studies were instrumental to validate the proposed binding mode, identifying His285 and Tyr316 as key residues
for ligand-dependent receptor activation. ITE analogues were also synthesized to develop a pharmacophoric model of relevant
interactions to AhR.
1
0
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